Cholesterol is a fundamental component of cellular membranes and plays a crucial role in regulating their structural properties. In addition, cholesterol serves as a precursor for important compounds, such as bile acids, steroids, and oxysterols. Although essential, elevated serum cholesterol is associated with heart disease and atherosclerosis. Notably, more than 38 million American adults have a total blood cholesterol e 240 mg/dL, which is considered high risk for cardiovascular disease. For these reasons, understanding how to maintain low, but sufficient, serum cholesterol levels is critical for human health. Cholesterol supply is controlled by multiple factors: dietary intake, synthesis, storage, catabolism and excretion. Cytochrome P450 monoxygenases play a key role in regulating cholesterol homeostasis through their functions in cholesterol, oxysterol, bile acid and steroid synthesis. Recently, we identified the heme-binding protein PGRMC1 as a binding partner and positive regulator of the cytochrome P450 Cyp51A1 in cholesterol synthesis. Using transfected cultured cells, we found that PGRMC1 binds tightly to a diverse set of P450 enzymes, including Cyp3A4, Cyp21A2 and Cyp7A1 that function in drug metabolism, steroid and bile acid synthesis, respectively. Based on these findings, we hypothesize that PGRMC1 is an important regulator of cholesterol homeostasis in animals. As a first step, we propose the following aims to test whether PGRMC1 is required for cholesterol homeostasis in mouse liver through regulation of Cyp51A1 and other P450 enzymes. AIM 1. To determine the function of PGRMC1 in liver cholesterol synthesis. AIM 2. To identify PGRMC1-interacting proteins from mouse liver. These experiments are a first step toward our long-term goal of understanding the function of PGRMC1 in systemic cholesterol homeostasis. The central role that cytochrome P450 enzymes play in regulation of serum cholesterol suggests that a clear understanding of PGRMC1 function will lead to improved therapeutic strategies for lowering serum cholesterol and preventing heart disease. PUBLIC HEALTH RELEVANCE: More than 38 million American adults have a total blood cholesterol e 240 mg/dL, which is considered high risk for cardiovascular disease. In this project, we will determine the function of the novel cytochrome P450 regulator PGRMC1 in systemic cholesterol homeostasis. The central role that cytochrome P450 enzymes play in regulation of serum cholesterol suggests that a clear understanding of PGRMC1 function will lead to improved therapeutic strategies for lowering serum cholesterol and preventing heart disease.